MBMB 451A Section One - Fall 2007

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Nucleosome

A.  Probing the structure of the histone octamer and chromatosome
	1.  General features
		a.  core histones: H2A, H2B, H3, and H4=octamer table
		b.  octamer +DNA = nucleosome core particle
		c.  nucleosomal core particle + linker histone = chromatosome
 		b.  linker histone (H1 H5, ...) can be removed by 0.5 M NaCl figure 20.3 (Genes VIII)
	2.  Dimensions of nucleosome stack 6 X 10 nm figure 20.5 (Genes VIII) and figure a, b, and c.
	3.  Most of the surface is covered with DNA (diameter of DNA is 2 nm)
		sites are brought close together that are 80 base pairs away
		entry and exit site sites are close together 
		figure 20.6 (Genes VIII)
	4.  DNase I footprinting and limit (complete) digestion	
		a.  nucleosomal ladder - native gel, >90% of  DNA is represented figure 20.8 (Genes VIII)
		b.  limit digestion gives fragment sizes of 146 base pairs figure 20.10 (Genes VIII)
		c.  core DNA (invariant 146 bps) vs. linker DNA (8 to 114 base pairs)
		d.  periodicity of 10 base pair cutting suggest B-form DNA
			divide core DNA up into 14 sites of ~10 bps each
			numbering system based on dyad symmetry
			slight variations of 10.7 base pairs of S4-S10 and S1-S4 and S10-S13 are 10.0 bps
				(typical B-DNA is 10.5 bps/turn)
			cutting is spread over 3-4 positions
			figure 20.13 (Genes VIII)
	5.  Supercoiling of DNA- caused by the DNA being restrained by wrapping of DNA
		b.  use minichromosome to measure extent of supercoiling due to nucleosome
		c.  to measure supercoiling due to nucleosome must first relax minichromosome
			by cutting and ligating with topoisomerase, then removing protein and measuring
			the supercoiling that is induced in free DNA
		d.  linking number paradox:  measurements indicate ~1 negative supercoil per nucleosome, 
			should be ~1.7-1.8 (# turns of DNA per nucleosome)

B.  Organization of histone octamer figure 20.22 (Genes VIII)
	1.  H3-H4 tetramer
		central core of octamer
		can organize DNA in vitro into particles that have some of the properities of nucleosome core particles
	2.  H2A-H2B dimer
		can cause to dissociate one dimer then another from octamer leaving the H3-H4 tetramer
			crosslinking studies show subunit-subunit interactions and protein-DNA interactions
		i.  protein-protein crosslinking
		ii.  protein-DNA crosslinking
	3.  Crystal structure of nucleosome core particle
		a.  histone fold - each subunit is interdigitated with another subunit ("handshake")
		b.  cylindrical wedge
		c.  tripartite - tetramer and 2 dimers
		d.  proteins form a superhelical structure or protein spool that corresponds well to DNA super helical path

 reference:  Nature 1997 Sep 18;389(6648):251-60 Crystal structure of the nucleosome core particle at 2.8 A resolution.
	4.  Placement of H1
		suggest that it binds to the linker DNA
		prevalent idea is that H1 seals the DNA in the nucleosome by binding DNA as it enters and leaves
	5.  Histone modifications - globular domain vs. flexible tails figure 20.26 (Genes VIII)
		a.  acetylation
		b.  methylation
		c.  phosphorylation
		d.  variant forms of H1

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Nucleic Acids  |  Methods-Nucleic Acids: Part A and Part B  and Molecular Cloning | DNA Supercoiling  |  Higher Ordered Structure  | Transcription: Part A and Part B  |  Chromatin Transcription  |  Regulation of Transcription  |  Course Material

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Last updated on August 30, 2007.